Herpes simplex virus complex

ABSTRACT

There is provided an HSV complex which comprises an avirulent HSV and a targeting agent which allows the HSV particle to infect and lyse a specific targeted cell. The inventors have found a way in which avirulent HSV can be targeted to disease cells, e.g. cancer cells, by incorporating an antibody binding domain into one or more viral glycoproteins.

The present invention relates to Herpes Simplex Virus (HSV) complex,including its production and its use. Particularly, but not exclusively,the invention relates to an HSV type I incorporating an antibody bindingdomain for targeting cells especially cancer cells.

HSV is an enveloped, icosahedral, double-stranded DNA virus that infectsmammals, including humans. Wild-type HSV infects and replicates in bothterminally differentiated cells and dividing cells. Wild type HSV isneurovirulent, entering the peripheral nervous system where active viralreplication is suppressed and the virus remains latent in neurones. HSVcan reactivate from the latent state to produce infectious lesions. TheHSV neurovirulence gene ICP34.5 is believed to condition post-mitoticcells (particularly neurones) for viral replication. (Thompson et al.Virology 172, 435-450 (1989); MacLean et al., J. Gen Virol. 72, 631-639(1991); Harland and Brown, J. Gen Virol. 66, 1305-1321 (1985); and Tahaet al., J. Gen Virol. 70, 705-716 (1989)). ICP34.5 deletion mutantscannot replicate in terminally differentiated cells but can lyticallyinfect dividing cells (Brown, S. M et al. 75, 2367-2377 (1994). TheHSV-1 mutant strain 1716 is an ICP34.5 deletion mutant (EP-B-0 571,410)of HSV type I strain 17, that has reduced virulence and greatly reducedlethality in mice, but it replicates as efficiently as wild type virusin actively dividing tissue culture cells (Maclean, A. R. et al. J. GenVirol. 72 631-639 (1991), Brown, S. M. et al, J. Gen Virol. 75 2367-2377(1994)). The ability of ICP34.5 deletion mutants to specifically targetand lyse dividing cells and not post mitotic cells makes them anattractive therapeutic agent for the treatment of cancer. HSV infectionof rapidly dividing cancer cells leads to death of the cells by lysis.The 1716 mutant kills tumour cell lines in tissue culture and, in arange of in vivo cancer models, the virus was shown to induce tumourregression and prolong survival (Kesari, S., Randazzo, B. P. andValyi-Nagy, T. Lab Invest. 73 636-648 (1995), MacKie E. A. et al. Br J.Cancer 74 745-752 (1996), Randazzo, B. P. et al. J. Invest Dermatol. 108933-937 (1997)). In clinical trials, direct injection of 1716 waseffective in treating patients with recurrent glioma (Rampling, R. etal. Gene Therapy 7 (10) 859-866 (2000)) and metastatic melanoma (MacKie,R. M., Stewart, B. and Brown, S. M. The Lancet 357 525-526 (2001)).

Significantly, in both of these trials there was no evidence for spreadof 1716 to surrounding normal tissue. A second HSV-1 ICP34.5 deletionmutant, G207, which additionally lacks the UL39 gene that encodes thelarge subunit of the viral ribonucleotide reductase, has also been shownto be safe and effective in patients with malignant glioma (Markert, J.M. et al. Gene Therapy 7 (10), 867-874 (2000)).

Although strains such as 1716 and G207 that have impaired neurovirulenceand that selectively infect dividing cells have strong therapeuticpotential for the treatment of human malignancies, some limitations oftheir use are anticipated by the inventors. 1716 is able to infect andkill a variety of tumour cells in tissue culture, but its permissiverange in vivo may be more restricted. For example, 1716 infection of Bor T cell lymphomas has not been reported. Additionally, the virus mayinfect tumour cells less efficiently in vivo than in cell culture. Theinventors have appreciated that it is desirable to overcome certaincell-type restrictions and increase the efficiency of infection oftumour cells so that modified HSV can be more widely, effectively andsafely applied as an in vivo therapy.

Broadly, the present invention provides a means of altering or modifyingthe tropism of HSV, so that a particular range of cell types can betargeted.

At its most general, the present invention provides an HSV complexcomprising a modified HSV and a targeting agent capable of targetingsaid modified HSV to a specific cell type, preferably a proliferatingcell e.g. a cancer cell. The invention further provides a method ofproducing the HSV complex and its use of the complex in the treatment ofdiseases such as cancer.

Thus, in a first aspect, the present invention provides an HSV complexcomprising an HSV linked to a targeting agent, capable of targeting aspecific cell type, where the genome of said HSV is modified in theterminal portion of R_(L) within Bam H1 s (0-0.02 and 0.81-0.83 mu).

The targeting agent is conveniently an antibody or component of anantibody, e.g. an antibody binding domain. The antibody is preferablycapable of specifically binding to a cell surface protein present of thecell type targeted. This is discussed below. The targeting agent ispreferably linked to the modified virus via a viral envelope protein sothat it is displayed on the surface of the virus. A convenient way ofachieving this is to form a fusion protein comprising the targetingagent and a viral envelope protein such as a glycoprotein.Alternatively, the targeting agent may be linked to the viral particleby chemical means, e.g. co-valently, or by a binding agent, e.g.avidin/strepavidin and biotin.

In a preferred embodiment, nucleic acid encoding the targeting agent isincorporated into the viral genome so that it is expressed as a fusionprotein with a viral envelope protein e.g. a glycoprotein, and as aresult displayed on the surface of the particle.

Thus, the invention provides an HSV which is capable of targeting aspecific cell type, said HSV lacking an expressible γ34.5 gene so as tolack neurovirulence and wherein the HSV expresses a targeting agent.

As an antibody binding domain forms a preferred embodiment of theinvention, the following description will concentrate on the use ofantibodies. However, it will be apparent to the skilled person thatother targeting agents may be used, e.g. members of a specific bindingpair such as a receptor and its ligand.

The antibody or antibody component incorporated into the viral envelopeinfluences the selectivity of the virus by enhancing the efficiency ofviral infection of a certain cell type or cell types. The HSV infectionprocess is initiated through contact between glycoproteins of the viralenvelope and glycoproteins of the target cell membrane. In the presentinvention, antibodies with specific affinity for membrane proteins ofthe chosen cell type are incorporated into the HSV viral envelope,increasing the affinity of the HSV for the surface of the chosen targetcell through the additional interaction between the antibody and thecell surface protein. The binding of the antibody binding domain to itstarget antigen on the cell surface will bring both virion and cellularmembranes into closer proximity and allow the viral envelopeglycoproteins to initiate fusion of the membranes, leading topenetration of the cell.

The HSV-1 virion envelope contains at least 10 integral membraneglycoproteins, several of which mediate entry into mammalian cells. Theinitial interactions between the virus and the cell are between viralglycoproteins gB and/or gC and cell surface heparan sulphateproteoglycans, but this interaction is insufficient for viralpenetration. Fusion of the viral and cellular membranes requires gD, gBand a gH/gL complex, and these proteins are presumed to act in concert.Specific receptor-mediated entry of HSV-1 involves interaction of gDwith HVEM/HveA (herpesvirus entry mediator A), a lymphotoxin receptorand member of the TNF receptor family. Expression of HveA in CHO cellsthat are normally refractory to viral penetration rendered thempermissive. Several other receptors have been identified usingnon-permissive CHO cells including the poliovirus receptor relatedproteins 1 and 2, now renamed HvecC and HvecB respectively. For thiswork, the inventors have been able to render a cell line normallyresistant to infection permissive for HSV-1 entry.

The present invention uses an HSV that has an impaired ability toinfect, replicate in or lyse terminally differentiated, non-dividingcells. In this form the virus is particularly suited for use as atherapeutic agent to treat diseases associated with proliferating cellssuch as cancer and non-cancer diseases such as Crohn's disease. Theinventors believe the modified virus in accordance with the presentinvention will be particularly useful in targeting proliferating T-cellsin cancer or non-cancer situations.

In a preferred embodiment, the HSV has been modified so that the geneencoding ICP 34.5 (gene γ34.5) is incapable of expressing a functionalgene product.

The modified virus preferably contains a modification in respect of thewild type virus within the Bam HI s region of the internal repeat R_(L)(0.81-0.83 mu) and within the counterpart region of the terminal R_(L)(0-0.02 mu) such that the modified virus (variant) lacks neurovirulence.

The modification of the virus genome may be achieved by deletion of oneor more nucleotides, insertion of additional nucleotides or any otheralteration of the nucleotide sequence such as rearrangement, orsubstitution. Preferably, the modification of the HSV genome is achievedby deletion of one or more nucleotides.

The HSV may be a spontaneously isolated deletion variant of the wildtype or it may be a wild type strain into which the desired modificationhas been introduced. Such modifications in the HSV may be made bygenetic manipulation, for example by site-directed mutagenesis, or byexcision of a portion of the genome with or without replacement with apre-prepared DNA cassette incorporating the required modification.

Preferably, the HSV is HSV type I (HSV-I) even more preferably HSV-1strain 17. In one embodiment, the HSV-1 strain will have a deletion ofat least 100 nucleotides in the Bam HI s' region between Alu I site at125074 nb and 125972 nb and within its counterpart sequence in TR_(L).

More preferably 0.5 to 3 kb of the Bam HI s' region and its counterpartin TR_(L) is deleted. Still more preferably about 0.7-2.5 kb is deleted.

Suitable modified HSV include HSV-1 mutant 1716 or G207. The productionof HSV1716 is described in EP 571,410-B which is incorporated herein byreference.

In addition to the above, the inventors have appreciated that, in orderto treat a diverse range of tumours, the HSV complex in accordance withthe invention will ideally have to be administered into the circulationof a patient. However, not only does the virus have to find the tumourcells (it can bind and be adsorbed by many different cell types) but italso has to contend with pre-existing immunological defences (e.g.neutralising antibodies) designed to eliminate the virus. Pre-existingimmunological defenses will be reasonably common as most people have hadprevious exposure to HSV-1. Given this, the present inventors haveappreciated that there is a need to develop a “stealth” virus thatavoids immunological detection and can specifically target tumour cells.Accordingly, the inventors have produced a stealth virus by eliminatingthe normal viral glycoproteins that mediate cell adsorption andreplacing them with antibody-directed entry mediating glycoproteinsincorporated into the virion structure. The principal viralglycoproteins involved in cell entry also provide the main neutralisingepitopes and their removal will minimise immunological activity againstsuch a virus. Thus, tumour antigen-directed HSV e.g. HSV1716 introducedinto the circulation can target many tumour types including disseminatedcancers that are either inaccessible or too numerous for directinjection or are too small to be detected.

Thus, the HSV complex as described above, e.g. HSV1716, that displays atumour specific targeting antibody in accordance with the first aspectof the present invention, may be modified such that the genes encodingviral glycoproteins essential for normal cell entry (e.g. principally gDbut also gC and/or gB, see below) are deleted or inactivated, therebyrendering the resulting virus dependent on tumour antigen/antibodyinteractions as the main route for cell infection. Deletion of theseglycoproteins from the virus particle also removes the principalneutralising epitopes and therefore greatly reduces immunologicaldefences when administered systemically.

Therefore, the HSV complex according to the first aspect of the presentinvention may be further modified so that one or more viralglycoproteins, e.g. gD, gC and/or gB, are inactivated or deleted suchthat they are unable to mediate entry of the viral particle into cells.It is preferable that the one or more glycoproteins are modified at thegenome level such that they cannot be expressed or cannot be expressedin a functional form. It is most preferable that the HSV genome ismodified so that the one or more gycoproteins cannot be expressed at allas this will provide the HSV variant with the additional advantage thatit can escape any pre-existing immunological defences in vivo.

As mentioned above, the various glycoproteins may be modified or deletedfrom the viral particle, preferably at a nucleic acid level. Themodification may include the incorporation of nucleic acid encoding thetargeting agent so that the targeting agent is expressed on the surfaceof the particle. Thus, the HSV genome may be modified, in addition tothe γ34.5 gene, such that one or more of the glycoproteins (e.g. gD, gCand/or gB) express the targeting agent, e.g. the antibody bindingdomain.

Preferably, the antibody or antibody component is specific for tumoursurface antigen, i.e. antigen found on the surface of a tumour cell andassociated with that cell, being either unique to tumour cells or beingmore abundant on tumour cells than on most if not all non-tumour cells.Many novel or a typical forms of normal proteins are expressed by tumourcells, and antibodies directed against these provide tumour targetingstrategies. For example, carcinoembryoinic antigen (CEA) is an importantmarker on many tumour cells and engineered antibodies directed againstCEA have undergone clinical trials (Mayer, A. et al., J. Immunol.Methods 231 261-273 (1999)). Engineered antibodies directed against theHer2/neu growth factor (Trastuzamab) and against CD20 (rituximab) havebeen licensed for the treatment of breast cancer and Non-Hodgkin'slymphoma respectively Holliger, P. and Hoogenboom, H. (1998), NatureBiotechnology 16, 1015. CD55 (decay accelerating factor) isover-expressed by tumour cells to block complement activation andantibodies directed against CD55 may have therapeutic potential (Li, L.et al. B. J. Cancer 84 (1) 80-86 (2001)). Incorporation of an antibodybinding domain that specifically targets tumour antigens such as CEA,Her2, CD20 and CD55 into the envelope of HSV will have the potential toalter its cellular tropism thus allowing infection of non-permissivetumour cells and possibly improving its ability to infect other tumourcells. For example, 1716 virions that display an antibody binding domainspecific for CD20 may be able to infect and kill B-cell lymphomas.

Further, HSV, e.g. HSV1716 virions that display tumour targetingantibodies and from which the normal HSV-1 entry glycoproteins aredeleted will only infect the targeted tumour cells.

The antibody binding domain may have specific affinity for a cellsurface protein found on the cell type from which the tumour originated,e.g. in the case of a glioma, the antibody or antibody componentincorporated into the HSV viral envelope would be specific for anantigen commonly associated with glial cells. The specificity of theavirulent HSV strain for infecting dividing cells would therefore befurther modified so that glial cells were preferentially infected by thevirus more than other types of dividing cells. By targeting dividingglial cells, the HSV should infect and lyse glioma cells moreefficiently than any other cells. The use of antibodies or antibodycomponents against particular cell types can also be used to extend thetropism of HSV to cell types that are not otherwise efficiently infectedby HSV, e.g. the use of antibodies or antibody components specific forantigen found on B cells would be expected to extend the tropism of HSVto B cells. Antibodies or antibody components of different specificitiesmay be included together in one HSV viral envelope. The combination ofthese specificities would be expected to give greater specificity oftargeting to the desired cell type.

The antibody binding domain would preferably be fused to an integralmembrane protein in the viral envelope, preferably an HSV glycoprotein.The preferred HSV-1 and HSV-2 glycoproteins are gB, gC and gD.

In a preferred embodiment, the antibody binding domain is in the form ofa single chain variable fragment (scFv).

In a second aspect, the present invention comprises a method of making amodified HSV complex according to the first aspect of the inventioncomprising the step of infecting a cell line that constitutivelyexpresses said fusion protein with an HSV, preferably a modified HSV,more preferably modified HSV-1.

In a third aspect, the present invention comprises a method of making amodified HSV complex according to the first aspect of the inventioncomprising the step of incorporating DNA encoding said fusion proteininto the viral genome of the modified HSV.

In a fourth aspect, the present invention comprises the use of amodified HSV complex according to the first aspect of the invention in amethod of medical treatment.

Preferably, the method comprises administering the HSV modified complexto a patient suffering from a disease associated with proliferation ofcells, e.g. cancer, wherein the HSV complex selectively lyses dividingcells.

There is also provided a pharmaceutical composition which comprises themodified HSV in accordance with the present invention and apharmaceutically acceptable carrier.

Pharmaceutical compositions comprising the modified HSV may beadministered intravenously or directly injected into a tumour orinfected site.

Aspects and embodiments of the present invention will now beillustrated, by way of example, with reference to the accompanyingfigures. Further aspects and embodiments will be apparent to thoseskilled in the art. All documents mentioned in this text areincorporated herein by reference.

FIG. 1. shows (a) the HSV-1 genome (with map units marked) in theprototype orientation; and (b) an expansion of BamHI k(s+q). The BamHI(B) and Alu (A) sites flanking the deletion in 1714/1716 are marked. Allcoordinates are based on the numbering of McGeoch et al. (1988). Alsoindicated are the positions of the 5′ end of IE1, the “a” sequence, theDR₁/U_(b) boudary in the “a” sequence, a 189 bp conserved open readingframe between HSV-1 and HSV-2 (R_(L) ORF) and the end points of the 759bp deletion in 1714/1716. The deletion extends from the DR₁/U_(b)boundary to remove the 5′ 107 bp of the R_(L) ORF.

FIG. 2. Construction of the vector pEL4. a) Sequence of the 5′ 99-meroligonucleotide used to PCR amplify scFv DNA and simultaneously insertan IgG VH leader sequence. The EcoRI site is underlined in red and theSfiI site is underlined in purple. The sequence between these two sitesencodes the IgG VH leader sequence that will target the expressedprotein to the secretory pathway of the cell. The sequence downstream ofthe SfiI site is from IgG VH. The 3′ primer was from IgG VL and insertsa NotI site allowing PCR amplification of scFV DNA with 5′ and 3′ SfiIand NotI sites respectively. Following PCR the scFv DNA with IgG VHleader was cloned into the plasmid pcDNA4A to give pEL4.

FIG. 3. Scale drawing of PCR-created N terminal deletions of HSV-1 gC.The full length gC protein is depicted at the top showing the signalpeptide (sp) and transmembrane domain (tm). The 14 gC deletions shownbelow are designated according to the amino acid at their new Nterminus. Thus Δ164gC has 163 amino acids deleted from its N terminus togive a gC fragment corresponding to amino acids 164-511.

FIG. 4. 1% agarose gel that shows PCR amplification of sequentiallydeleted gC DNAs (lanes 1-14). M=DNA ladder with the location of the 200bp and 1000 bp bands indicated. A common 3′ PCR primer, corresponding tothe C-terminus of gC but without the stop codons inserts an XbaI siteand allows cloning in-frame with the vector myc and 6-his tags. The 5′primers used were randomly selected from the gC coding sequence to givesequential deletions of between 6-90 nucleotides. The 5′ primer insertsa NotI restriction site that allows the gC DNA to be cloned in-framewith the scFv DNA in pEL4. NotI/XbaI digested PCR amplified DNAs werecloned into pEL4 digested also with these enzymes to give scFv/gC fusionprotein expression constructs.

FIG. 5. Western blots that show expression of scFv/gC fusion proteins inwhole cell extracts and their incorporation into HSV1716 virusparticles. Lanes 5-8, 9-11, 16 and 17 are whole cell extracts. Lanes1-4, 12-15 and 18 are virus preparations. Lanes 1 and 5=Δ193gC, lanes 2and 6=Δ215gC, lanes 3 and 7=Δ251gC, lanes 4 and 8=Δ328gC, lanes 9 and13=Δ410gC, lanes 10 and 14 =Δ424gC, lanes 11 and 15=Δ362gC, lane12=Δ457gC, lanes 16-18=Δ227gC. Lanes 19 and 20 show mock viruspreparations made from cell lines expressing Δ227gC and Δ424gCrespectively, no bands were detected in these samples. Note that thecell line expressing Δ251gC gives a strong band in the cell extract(lane 7) but shows only a weak band in the virus preparation (lane 3).In contrast to this, Δ328gC is expressed at intermediate levels in thecell line but gives a strong band with its virus preparation. Theoverall results are summarized in Table 1.

EXAMPLE 1 Method of Making HSV-1 1716 Strains wherein Fusion ProteinsBetween Anti-Tumour scFvs and Envelope Glycoproteins B, C and D areIncorporated into the Virion Envelope

Recombinant scFv variants of monoclonal antibodies that bind differentextracellular epitopes of CD55 are derived by standard protocols (seePope, A. R., Embleton, M. J. and Mernaugh, R. (1996) In AntibodyEngineering (eds McCafferty, Hoogenboom and Chiswell) Practical ApproachSeries, Oxford University Press Inc., New York. 1-40). scFv areparticularly suitable for incorporation into the fusion protein becausethey are encoded by a single nucleotide sequence. scFv can beconveniently engineered using recombinant antibody technology(Hoogenboom, H. R. and Chames, P. Immunology Today 21 (8) 371-377(2000)). Recombinant antibodies are predominantly produced using mRNAisolated from hybridomas or from populations of lymphocytes isolatedeither from the spleens of immunised animals or from human peripheralblood. DNA fragments encoding immunoglobulin heavy (V_(H)) and light(V_(L)) chain binding sites are amplified separately by RT-PCR and fusedto produce fragments encoding single chain antibody molecules (thescFv). The scFv polypeptide effectively recreates the antigenrecognition site in a single protein that retains high affinity binding.The cloned scFv can readily be genetically fused with the domains ofother proteins, for example, with the M13 gIII coat protein for displayon phages or to the enzyme carboxypeptidase G2 for antigen directedenzyme prodrug therapy (ADEPT).

Following RT-PCR cloning, sequencing and linkage of the antibody VH andVL for each monoclonal antibody, the scFv-encoding DNAs will beamplified using PCR primers that incorporate SfiI and NotI restrictionenzymes sites at the 5′ and 3′ ends respectively for cloning in thephagemid vector pHEN2 or for construction of fusion proteins. The choiceof restriction enzyme sites will depend on the sequences of the scFv andglycoproteins used. E. coli HB2151 will be transformed with the phagemidvectors and scFvs expressed by IPTG induction. ScFv expressed from pHEN2have c-myc and 6-his tags for purification/detection. The reactivitiesof the recombinant scFv will be compared with their respectivemonoclonal counterparts by Western Blotting and FACS analysis using CHOcells stably transfected with plasmid that expresses their targetantigens.

DNA encoding an scFv with an IgG VH leader sequence was cloned into theplasmid pcDNA4 (Invitrogen) to create the vector pEL4 (FIG. 2). The 5′primer used to amplify the scFv DNA incorporates an IgG VH leadersequence, linked to the scFv DNA by a SfiI site. The primer also insertsan EcoRI site 5′ to the IgG VH leader sequence. The scFv DNA can beremoved and replaced by alternative scFv DNAs using SfiI/NotI digestion.The leader sequence can be removed by EcoRI/SfiI digestion and replacedwith other leader sequences, eg gC signal peptide sequences. Such leadersequences with EcoRI/SfiI restriction sites can be obtained either byPCR using appropriate primers or by using chemically synthesisedcomplementary oligonucloetides.

A number of HSV-1 strain 17⁺gB, gC and gD DNA fragments are PCR-clonedfrom the viral genome using methods which have previously beensuccessful for other herpesvirus proteins (Sun, Y. and Conner, J. (1999)The U28 ORF of human herpesvirus-7 does not encode a functionalribonucleotide reductase R1 subunit. Journal of General Virology 80:2713-2718. Sun, Y. and Conner, J. (2000) Characterisation ofhetero-subunit complexes formed by herpes simplex type 1 and equineherpes virus type 4 ribonucleotide reductase R1 and R2 subunits. BiochemJ. 347, No 1: 97-104. Incorporated herein by reference. The primers usedto amplify the DNAs will incorporate appropriate restriction enzymesites for fusion to the scFv DNA and cloning into pEL4. PCR primers foramplification of glycoprotein DNA will be designed such that a series ofrandom, sequentially deleted N-terminally truncated proteins areexpressed, each deletion will remove approximately 2-30 amino acids upto the region encoding the transmembrane domain. For example, gCcomprises 511 amino acids with the transmembrane region locateddownstream of amino acid 479, a family of 14 sequentially deletedN-terminally truncated polypeptides (FIG. 3) for fusion to scFvs havebeen cloned. Examples of PCR-amplified gC DNAs are shown in FIG. 4. Theprimers used for PCR amplification of the gC DNA fragments incorporatedNotI and XbaI sites at the 5′ and 3′ ends respectively. PCR-amplifiedDNA was digested directly with the appropriate enzymes (i.e. NotI/XbaIfor gC fragments) and cloned into pEL4 digested also with these enzymes.The resulting constructs express scFv/gC fusion proteins with C-terminalmyc and 6-his tags, under control of the CMV IE promoter. The promoterand tags are provided by the pcDNA4 backbone of pEL4 as is a zeocinresistance gene that allows production of stable cell lines usingantibiotic selection. DNA fragments were also cloned into thePCR-cloning vector pGEM-T Easy and sequenced to ensure that noPCR-induced mutations have been incorporated.

BHK cells were transiently transfected with each of the pEL4 constructsusing lipofectamine and, after 48 hrs, zeocin selection (10 ug/ml) wasinitiated. Cells were selected with zeocin for approximately 21 days andextracts prepared for Western blotting. For each cell line, apolypeptide of the appropriate molecular size for the scFv/gC fusionprotein was detected with the anti-myc tag monoclonal antibody 9B11 (NewEngland Biolabs).

Examples of expression are shown in FIG. 5 and estimates for the levelsof expression in each of the cell lines using the Western blot data arepresented in Table 1. Immunofluorescence using 9B11 and an anti-murineIgG/FITC conjugate demonstrated a perinuclear/Golgi localisation for allof the expressed fusion proteins. Incorporation of scFv/glycoproteinfusion proteins into the HSV1716 envelope using stably transfected BHKexpressing cell lines was analysed by Western blotting with 9B11.HSV1716 at 10 pfu/cell was used to infect each of the cell lines andvirus harvested from the culture medium 24-28 hours later was analysedby Western blotting with 9B11. Examples are shown in FIG. 5 and resultssummarised in Table 1. No myc-tagged proteins were detected in similarpreparations made from mock-infected cells. Most scFv/gC fusion proteinswere incorporated into the virus (Table 1). Some were more efficientlyincorporated than others (eg Δ328gC and Δ457gC are expressed atintermediate levels in their respective cell lines but are present asstrong bands in their virus preparations) whereas incorporation ofothers expressed at high levels in their cell line was poor (eg Δ251gC).

Recombinant viruses expressing the most appropriate scFv/glycoproteinfusions are created using a variant of 1716 that expresses greenfluorescent protein (GFP). Incorporation of scFv/glycoprotein fusionsare confirmed as described above.

EXAMPLE 2 Infection of BHK Cells by HSV-1 IncorporatingscFv-Glycoprotein Fusion Proteins

The ability of the viruses incorporating ScFv-glycoprotein fusion (asproduced in example 1) to infect BHK cells is analysed by single-stepgrowth experiments and compared with 1716. The tropism of thesescFv-glycoprotein fusion viruses is investigated using CHO cells thatconstitutively express CD55. Penetration of the viruses into CHO cellsis confirmed using GFP reporter gene analysis.

Results in CHO cells will identify the factors that allowantibody-mediated viral infection and recombinant viruses will becreated by cloning the appropriate scFv/glycoprotein DNA into the viralgenome. The genomes of these viruses can be further modified by deletionor inactivation of glycoprotein genes such as gD, gC and gB using forexample, homologous recombination.

By accumulating monoclonal antibodies to tumour specific antigens suchas CEA or CD20, it is then possible to construct a panel of oncolyticherpesviruses with improved targeting to the tumour of choice. TABLE 1Expression in cells and incorporation into HSV1716 of scFv/gC fusionproteins. Incorporation into fusion protein Expression in cells virusΔ164 gC ++ − Δ193 gC ++ ++ Δ215 gC ++ ++ Δ227 gC +++ ++ Δ251 gC +++ +Δ270 gC − − Δ276 gC +++ +++ Δ313 gC + + Δ326 gC − − Δ328 gC ++ +++ Δ362gC ++ ++ Δ410 gC +++ +++ Δ424 gC +++ +++ Δ457 gC ++ +++Expression in cells and incorporation into virus were determined byWestern blotting.+++ = strong reactivity,++ = intermediate,+ = weak and − = undetected

1. An HSV complex capable of targeting a specific cell type, saidcomplex comprising an HSV modified in the terminal portion of R_(L)within BamH1 s (0-0.02 and 0.81-0.83 mu) so as to lack neurovirulence,and a targeting agent linked to the modified HSV, wherein said targetingagent comprises an antibody binding domain.
 2. (canceled)
 3. An HSVcomplex according to claim 1 wherein said antibody binding domain is inthe form of a Single Chain Variable fragment (ScFv).
 4. An HSV complexaccording to claim 1 wherein said targeting agent is an antibody.
 5. AnHSV complex according to claim 1 wherein the targeting agent is capableof specifically binding to a cell surface protein of the cell typetargeted.
 6. An HSV complex according to claim 5 wherein the cellsurface protein is a tumour associated antigen.
 7. An HSV complexaccording to claim 6 wherein the tumour associated antigen is CEA, Her2,CD20 or CD55.
 8. An HSV complex according to claim 1 wherein thetargeting agent is linked to the modified virus via a viral envelopeprotein.
 9. An HSV complex according to claim 8 wherein the targetingagent and the viral envelope protein form a fusion protein.
 10. An HSVcomplex according to claim 8 wherein the viral envelope protein is anHSV glycoprotein.
 11. An HSV complex according to claim 1 wherein theHSV has been modified such that gene γ34.5 is incapable of expressing afunctional product.
 12. An HSV complex according to claim 1 wherein theHSV is HSV-I.
 13. An HSV complex according to claim 1 wherein the HSV isHSV-I strain
 17. 14. An HSV complex according to claim 1 wherein the HSVis HSV1716.
 15. An HSV which is capable of targeting a specific celltype, said HSV lacking an expressible γ34.5 gene so as to lackneurovirulence and wherein said HSV expresses a targeting agent, whereinsaid targeting agent comprises an antibody binding domain.
 16. An HSVaccording to claim 15 wherein said targeting agent is expressed as afusion protein with a viral glycoprotein.
 17. An HSV according to claim15 further comprising the additional modification of one or more viralglycoproteins.
 18. An HSV according to claim 17 wherein the additionalone or more viral glycoproteins are deleted.
 19. An HSV according toclaim 17 wherein the additional one or more viral glycoproteins aremodified by incorporation of the targeting agent.
 20. An HSV accordingto claim 17 wherein the additional glycoproteins are gD, gC and/or gB.21. (canceled)
 22. An HSV according to claim 15 wherein said antibodybinding domain is in the form of a Single Chain Variable fragment(ScFv).
 23. An HSV according to claim 50 wherein the cell surfaceprotein is a tumour associated antigen.
 24. An HSV according to claim 23wherein the tumour associated antigen is CEA Her3, CD20 or CD55
 25. AnHSV according to claim 15 wherein the HSV is HSV-I.
 26. An HSV accordingto claim 25 wherein the HSV is HSV-I strain
 17. 27. An HSV according toclaim 26 wherein the HSV is HSV1716.
 28. (canceled)
 29. (canceled)
 30. Amethod of producing an HSV according to claim 15 comprising: (a)modifying the HSV genome so as to prevent γ34.5 gene from expressing afunctional protein; (b) modifying the HSV genome by incorporatingnucleic acid encoding a targeting agent so that said targeting agent isexpressed as a fusion protein with a viral coat protein, wherein saidtargeting agent comprises an antibody binding domain; (c) expressingsaid modified HSV in a cell.
 31. (canceled)
 32. A method according toclaim 30 wherein said antibody binding domain is in the form of a SingleChain Variable fragment (ScFv).
 33. A method according to claim 30wherein said targeting agent is an antibody.
 34. A method according toclaim 30 wherein the targeting agent is capable of specifically bindingto a cell surface protein of the cell type targeted.
 35. A methodaccording to claim 34 wherein the cell surface protein is a tumourassociated antigen.
 36. A method according to claim 35 wherein thetumour associated antigen is CEA, Her2, CD20 or CD55.
 37. A methodaccording to claim 30 wherein the targeting agent is linked to themodified virus via a viral envelope protein.
 38. A method according toclaim 37 wherein the targeting agent and the viral envelope protein forma fusion protein.
 39. A method according to claim 37 wherein the viralenvelope protein is an HSV glycoprotein.
 40. A method according to claim30 wherein the HSV is HSV-I
 41. A method according to claim 30 whereinthe HSV is HSV-I strain
 17. 42. A method according to claim 30 whereinthe HSV is HSV1716. 43-47. (canceled)
 48. A pharmaceutical compositioncomprising an HSV complex according claim 1 and a pharmaceuticallyacceptable carrier.
 49. A pharmaceutical composition comprising an HSVcomplex according to claim 15 and a pharmaceutically acceptable carrier.50. An HSV according to claim 15 wherein the targeting agent is capableof specifically binding to a cell surface protein of the cell typetargeted.